Molecular cloning of a mink prion protein gene ... Recent experiments have shown that the prion protein. (PrP), which, in its ..... Cloning: A Laboratory Manual.
Journal o f General Virology (1992), 73, 2757-2761.
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Molecular cloning of a mink prion protein gene H. A. Kretzschmar, l*~ M. Neumann, 1 G. Riethmiiller 2 and S. B. P r u s i n e r 3 Institutes of 1Neuropathology and 2immunology, University of Munich, Germany and 3Departments of Neurology, Biochemistry and Biophysics, University of California, San Francisco, California, U.S.A.
Transmissible mink encephalopathy (TME) is a rare disease which is presumably transmitted to ranchraised mink from scrapie-infected sheep offal or bovine spongiform encephalopathy-infected cattle products. Although the infectious agent of T M E has not been isolated, there is circumstantial evidence that T M E is caused by prions. The experimental host range of T M E includes sheep, cattle, monkeys and hamsters. However, T M E has never been transmitted to mice. Since experiments in transgenic animals have shown
that the prion protein (PrP) gene modulates the susceptibility, incubation time and neuropathology of prion-induced disease, we have started to analyse the mink PrP gene. PrP, as deduced from a genomic D N A sequence, consists of 257 amino acids and overall shows similarity of 84 to 90 % with the sequences of the PrPs of other mammalian species. It remains to be determined whether these differences in the primary structure of PrP will explain the peculiar host range of TME.
Transmissible mink encephalopathy (TME) is a rare disease of ranch-raised mink which was first reported in Wisconsin in 1947. Since that time, only 22 additional outbreaks have been recorded world-wide (Marsh, 1991). The clinical, pathological and biochemical features of TME suggest that it is a prion disease very similar to scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in mule deer and elk, and Creutzfeldt-Jakob disease, kuru and the Gerstmann-Str/iussler-Scheinker syndrome in humans (Marsh & Hanson, 1969; Kimberlin & Marsh, 1975; Marsh & Kimberlin, 1975). Epidemiological studies have associated the occurrence of TME with the feeding of contaminated food items, presumably scrapie-contaminated sheep offal. Recent epidemiological findings (Marsh et al., 1991) suggest that the disease may be transmitted to mink from BSE-infected fallen cattle. Experimentally, TME is readily transmissible to ferrets, squirrel monkeys, cattle and hamsters. In contrast to BSE, TME has not been shown to be transmissible to mice, and scrapie is not transmissible to mice after mink passage (Marsh et al., 1991). Recent experiments have shown that the prion protein (PrP), which, in its scrapie isoform (prpSc), co-purifies with the infectious agent of prion diseases, the prion, has a normal cellular isoform (PrP c) which plays a crucial role in the determination of incubation time, the
specificity of pathological changes and species barriers (Westaway et al., 1987; Scott et al., 1989). In addition, mutations and insertions in the PrP gene have been linked to inheritable human prion diseases (Hsiao et al., 1989; Owen et al., 1989), and transgenic animals expressing one of these mutations (Leu for Pro at codon 101 in the mouse) spontaneously develop neurodegeneration and consequently die of a disease very similar to scrapie (Hsiao et al., 1990). To elucidate the role mink PrP might play in the pathogenesis and transmission of TME, we cloned and analysed the mink PrP gene. R N A from mink brain tissue was prepared by a CsCI gradient procedure (Chirgwin et al., 1979). Total R N A (10 ktg) was separated on a formaldehyde-agarose gel and transferred to a Hybond-N membrane (Amersham). R N A on the filters was hybridized to an 864 bp fragment of the human PrP gene containing the entire open reading frame (ORF) (Hsiao et al., 1989), and labelled by random priming with [~-32p]dATP (Feinberg & Vogelstein, 1983). Hybridization was performed in 1% BSA, 7% SDS, 0.5 M-Na2HPO4 and 1 mM-EDTA. The membranes were washed at 65 °C in 3 x SSC, 10 mMNaH2POJNa2HPO4, 10 x Denhardt's solution and 5% SDS, followed by a 10 min wash in 1 x SSC, 1% SDS. Mink D N A was prepared from liver tissue by a modified proteinase K procedure (Gross-Bellard et al., 1973). For Southern blot analysis, 10 ~tg D N A was digested with BamHI, BgllI or EcoRI, run through a 0.7% agarose gel and transferred to a Hybond-plus membrane (Amersham). Hybridization and washing conditions were identical to Northern blot conditions.
t Present address: Department of Nearopathology, University of G6ttingen, Robert-Koch-Strasse 40, D-3400 G6ttingen, Germany. 0001-0915 © 1992 SGM
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Mink liver D N A was partially digested with Sau3A and size-fractionated on a sucrose gradient as described by Maniatis et al. (1982). A genomic D N A library was prepared using BamHI-digested 2 phage EMBL3. Phage plaques were screened with the 32p-labelled 864 bp fragment of the human PrP gene (see above); hybridization was for 12 h at 65 °C. Hybridization and washing conditions were as described for Northern and Southern blotting. Approximately 400000 phage plaques were screened, one of which gave a positive hybridization signal. The insert was cloned into a plasmid (pMa) and subsequently sequenced following a variation of the dideoxynucleotide chain termination method (Chen & Seeburg, 1985) using a T7 sequencing kit (Pharmacia) and specially synthesized oligonucleotides. Southern blot analysis was used to detect PrP genes in a number of mammalian species (Westaway & Prusiner, 1986). Cloning and sequencing has shown that in all species analysed, the PrP gene is a single copy gene with an uninterrupted protein-coding ORF in one exon. Southern blot analysis using a human PrP D N A probe containing the entire PrP ORF revealed single bands after digestion of mink genomic D N A with BamHI, BgllI or EcoRI (Fig. 1). These findings are compatible with a single compact PrP gene in mink. PrP mRNAs from different species show considerable variation in size, ranging from 2 kb to 4.6 kb (Westaway et al., 1987; Goldmann et al., 1990). Using a human PrP D N A probe containing the entire ORF in Northern blot analysis of total brain RNA, we estimated the size of mink PrP m R N A to be 2-3 kb (Fig. 2), within the size range of rodent and human PrP mRNAs. A genomic mink D N A library was prepared from mink liver DNA, plaques were screened for PrP-related sequences by hybridization to a human PrP gene probe and a positive clone was isolated. Large parts of the mink PrP gene, including the coding region and 3' untranslated region, were sequenced. The sequenced ORF consists of 771 nucleotides, followed by a putative 3' untranslated sequence of approximately 1650 nucleotides. The nucleotide sequence surrounding the A U G initiation codon (ATCATGG) is consistent with the consensus sequence (ANNATGG) for eukaryotic initiation sites (Kozak, 1983). At position 772, after codon 257 of this ORF, there is a stop codon (TGA), which is followed by a long trailer with the possible polyadenylation signals TATAAA at positions 2305 and 2316 and ATTAAA at position 2342. Both are variants of the consensus sequence (AATAAA) preceding the polyadenylation site of most eukaryotic mRNAs (Proudfoot & Brownlee, 1976), which are also found in the human 3' untranslated region. The deduced mink PrP sequence consists of 257 amino acids and has an Me of 28941 prior to post/
(a)
Fig. 1. (a) Southern blot analysis. High M r DNA from mink liver was digested with BamHI, BgllI or EcoRI (lanes 1 to 3), electrophoresed through a 0.7~ agarose gel and Southern-blotted. Hybridization was performed with a human 864 bp fragment containing the entire coding region of the human PrP gene. (b) Northern blot analysis of brain RNA. Lane 1, mouse; lane 2, mink. Total mouse and mink brain RNA (10 ~tg) was analysed in lanes 1 and 2, respectively. Hybridization was performed with the human 864 bp fragment. The positions of 18S and 28S RNAs are indicated.
translational modifications. A region of 24 amino acids at the N terminus is typical of a signal peptide (von Heijne, 1985), with a hydrophobic core (LLVLFVA) and a small uncharged residue (C) at the putative signal sequence cleavage site. We predict that the mature protein commences at the lysine residue at codon 25, and, before post-translational modification, has an Mr of 25 816. There are two possible asparagine-linked glycosylation sites at positions 185 and 201, and there is also an extremely hydrophobic C-terminal sequence. Southern blot analysis has revealed a single copy PrP gene in a number of mammalian species. The proteincoding region has been found to be located in one uninterrupted ORF in one exon in all species investigated. Southern blot and sequence analysis of genomic mink D N A shows that the organization of the mink PrP gene is identical to that of other mammalian species. We have deduced that the mink PrP consists of 257 amino acids, but as only one genomic clone was sequenced, no information is available about possible
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.1 ATG GTG AAA AGC CAC ATA GGC AGC TGG CTC CTG GTI CTC TTT GTG GCC MET Va[ Lys Set His l i e Gly Ser Trp Leu Leu Va[ Leu Phe Vat Ala 1 15 .100 AAG CCT GGA GGA GGC TGG AAC ACT GGG GGG AGC CGA TAC CCA GGG CAG GGC Lys Pro G[y Gly GLy Trp Asn Thr G[y Gly Ser Arg Tyr Pro Gty Gin Gty 45 3O .200 GGC GGC TGG GGC CAG CCC CAC GGG GGT GGC TGG GGA CAG CCC CAC GGG GGT Gly Gty Trp G[y G[n Pro His Gly G[y Gly Trp Gly Gin Pro His G[y Gty 6O .300 CCG CAT GGT GGC GGT GGC TGG GGT CAA GGT GGT GGG AGC CAC GGT CAG TGG Pro His Gly G[y Gly Gly Trp G[y Gin G[y Gty G[y Ser His G[y Gin Trp
TCATTTTGTTTTGTTTTGTTTTGTTTGCAGATAAGCCATC
ACA TGG AGT GAC ATT GGC TTC TGC AAG AAG CGG CCA Thr Trp Ser Asp l i e Gly Phe Cys Lys Lys Arg Pro .150 AGT CCT GGA GGC AAC CGC TAC CCA CCC CAG GGT GGT Ser Pro G[y G[y Asn Arg Tyr Pro Pro Gin G[y Gly
.250 GGC TGG GGT CAG CCC CAC GGG GGT GGC TGG GGA CAG G[y Trp Gly Gin Pro His Gly G[y G[y Trp Gty Gin 75 GGC AAG CCC G[y Lys Pro 105 .400 ATG CTG GGG MET Leu Gly 135
AGT AAG CCC AAA ACC AAC ATG AAG CAT GTG Ser Lys Pro Lys Thr Asn MET Lys His Va[
AGC GCC ATG AGC AGG CCC CTC ATT CAT TTT Ser ALa MET Ser Arg Pro Leu ]le His Phe
.500 CCC AAC CAA GTG TAC TAC AAG CCG GTG GAT CAG Pro Asn Gin Va[ Tyr Tyr Lys Pro Va[ Asp Gin 165 .600 CAC ACG GTG ACC ACC ACC ACC AAG GGC GAG AAC His Thr VaL Thr Thr Thr Thr Lys Gly GLu ASh 195
ACC CAG TAC CAG CGA GAG Thr Gin Tyr Gin Arg Gtu 225 .750 TCA CTG CTC ATT CTC CTG Ser Leu Leu I r e Leu Leu
90 .350 GCG GGA GCC GCA GCA GCC GGG GCG Ata G[y A[a Ata A[a Ala Gty Ala 120 .450 GGC AAC GAC TAT GAG GAC CGC TAC G[y Asn Asp Tyr G[u Asp Arg Tyr 150
GTC GTG GGG GGC CTG GGC GGC TAC Vat Val Gty G[y Leu Gty Gty Tyr
TAC CGT GAG AAC ATG TAC CGC TAC Tyr Arg G[u Asn MET Tyr Arg Tyr
.550 TAC AGC AAC CAG AAC AAC TTC GTG CAT GAC TGC GTC AAC ATC ACG GTC AAG CAG Tyr Ser Asn Gln Asn Asn Phe Vat His Asp Cys VaL Asn I r e Thr Va[ Lys Gin 180
.650
TTC ACG GAG ACC GAC ATG AAG ATC ATG GAG CGC GTG GTG GAG CAG ATG TGT GTC Phe Thr Glu Thr Asp MET Lys ILe MET GLu Arg VaL Val Glu Gin MET Cys Vat 210 .700 TCC GAG GCT TAC TAC CAG AGG GGG GCG AGC GCC ATC CTC TTC TCG CCC CCT CCC GTG ATC CTC CTC ATC Ser Glu Ala Tyr Tyr Gin Arg Gty Ala Ser Ata lie Leu Phe Ser Pro Pro Pro Va[ l i e Leu Leu l i e 240 .800 ATA GTG GGA TGAGGATGGCCTTCCCATTCTCTCCATCGTCTTCACCTTTTACAGGTTGGGGGAGGGGGTGTCTACCTACAGCCCTGTA Ile Val GLy 255 .900
GTGGTGGTGTcTcATTccTGcTTcTcTTTATcACCcATAGGCTAATccccTTGGcccTGATGGCCCTGGGAAATGTAGAGcAGACccAGGATGcTATTTATTcAAGcccCcATGT .1000 GTTGGAGT•cTT•AGGGG••AATG•TAGTGCAGGG•TGAGAATAA•AGcAAAT•AT•ATTGGTTGAc•TAGGG•TGcTTTTTTGTTGTTGTTGTcTAGTGcAGcTGAccGAGGcT .1100 AAAA•AATT•TcAAAACAGTTTT•AAATACCTTTG••TGGAAA••T•TGG•T••TGCTGCAGCTAGAGCT•AGTACATTAATGT••CATCTTAGC•GTGT•TTCATAGCAA•TTG .1200 .1300 GGGAAGTTTTTcTc•••ACT•TAAAAGAA•GCGATTGCA•TT••CTGTG•AAAGAACATTTcTG••AAATTTGAAAGGAGGCCACATGATATTCATTCAAAAAGCAAAA•TAGAA .1400 ACC•TTTGCTCTTGGACGCAAGCCCGGCCTGCTAGGAGCACCAAACTGGGGCGATGGTTTGCATTCTGCGGCGTGGGCTATGCGGCAGCCGAGGTGTCCAGCGTAAATATTGATG .1500 CGAC••TAGACCTAGGCAGAGGATGTTTGCACAGGGAATGAACATAATCAACAGTG•GAAAATGCTACAAAAAATCCCACACTGGGGAGCAGTGTCCTTGGAGGCAAGTTTTTTT .1600 CCTTTTGGGACATTTAAAG•CC•TATATGTGGCATTCCTTTCTTTCGTAACCTAAACTATAGATAATTAAGG•AGTTAAAAATTGAACTTCCTTCCAGGCCCCAAGAGCAAATCT .1700 TTGTTCACTTACCTGGAAACCAGAATGATTTTGACACAGAGGAAGGTGCAGCTGTTAAAATAACCCTCATCCTAGAAGATTGCATCATGGAGAAAACGATCCGTAGACAAAAATG
.1800 AT~G~ATTT~TT~ATTGC~GT~T~GTAATTGA~AGAAA~AGAATTATGT~AAGT~TAGTTT~TATAAT~AG~TTTTGAAT~AAAGAATGGAAGT~AT~AAAAA~G .1900 .2000 AAATACCTTAGGTCACCCATGACAGAAATACCCATT•AGGTTAGAAAAAAGGAATTCTGTTAACTGTTATTTAAGTAAGGcAAAATTATTGT••GGATTGTTCGATAT•ATCAGc .2100 TAGCAGATAAATTAGCATTCTGCAATGTTCC•GGCTTGCACTGTGCGGGTATTTGATGTTAAAAAAAATTATTATATATATTGTGTATGACAAACTTAGAAGTTTTTGCTAGAGG .2200 AGTTAACAT•TGATATATCTAAT•CACCACCAGTTTTGGAAGGTACTAAATA•TTAATATGTAGAAAT•CTTTTGCGTGGTC•TCAGG•TTA•ACGTGcA•TGAATAGTTTTGTA .2300 TGATAGAG•c•ATGTGGT•TT•GAAATATG•ATGTA•TTTATATTTT•TATATTTGTAAcTGGG•ATGTA•TTGTATAAAAAATGTATAAACATT•GAA•T•TTGA•TAGAATTA .2400 AACAGGAACTGAGTGTGTCCCATGTGTTTGCAGTGACATTCACCACCGCACCCTGTGTTGG
Fig. 2. Nucleotidesequence ofthe putative coding region and the 3'untranslated region o f t h e m i n k PrP gene with the deduced amino acid sequence.
mink PrP polymorphisms. The deduced protein shows 84 to 95~o overall similarity with the PrPs from other species. Genomic D N A sequence similarity ranges from 81 ~o, when compared to mouse, to 87~, when compared
to cattle. At the amino acid level, the greatest similarity is also observed between the mink and sheep PrP sequences (Table 1). The structure of the 3' untranslated region is most closely related to that of the human trailer.
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1
25
50
75
I00
Human MANLGCWMLVLFVATWSDLGLC KKR PKPGG-WNTGGS RYPGQGS PGGNRY P P Q ~ w C - Q PHGGGWGQ PHC-GGWC-QPHGGGWGQ PHGGG-WGQGGGTHSQWN Hams ter SYLA MTV T N Mouse YLA TMTV -T S S N MVKSHI S L I F G G S G G Mink Rat ............................ S T - S N MVKSHI S I M V G G -S S Sheep M V G G - G Cattle MVKSHI S I
Human Hamster Mouse Mink Rat Sheep Cattle
125 150 175 200 • . . . KPSKPKTNMKI~MAGAAAAGAVVGGLGGYMLGSAMSRPII HFGSDYEDRYYR ENMHRy pNQVYYR PMDEYSNQNNFVHDCVNI TI KQHTVTTTTKGENFTE MM N W N VQN L V M N W Y vQ V L N Y KVQ V b V ML N W Y VQ V L N Y VR V V L VQ VE 225
Human Hamster Mouse Mink Rat Sheep Cattle
250
TDVK)~4ERVVEQMCITQYERESQA~-QRGSS-MVLFSSPPVILLISFLIFLIVG I I T QK DG R -A M V QK DG R ST M I v Q E A -AI P L L v QK DGR -A I I Q A -Vl I Q A -Vl
Fig. 3. Comparison of the mink PrP sequence with the human (Kretzschmar et al., 1986), hamster (Oesch et al., 1985; Basler et al., 1986), mouse (Locht et al., 1986; Westaway et al., 1987), rat (Liao et al., 1987), sheep (Goldmann et al., 1990) and bovine (Goldmann et aL, 1991) sequences.
Table 1. Similarity between the m i n k P r P sequence a n d other k n o w n P r P sequences
PrP Mouse Hamster Cow Sheep Human
Nucleotide identity (~)
Amino acid identity (%)
Amino acid identity (excluding signal peptide) (~)
81-19 82.36 86.64 87-03 84-69
84-43 84.82 93-00 93.77 87.55
87-98 88.41 93.99 94-85 89.70
Both are approximately 1650 bp in total length and the similarity is 7 5 ~ . The putative signal sequence of 24 amino acids is most closely related to the signal sequence in sheep and cattle PrPs, with a stretch of five identical amino acids [(M/V)KSHI] found only in these three species. Two glycine codons are inserted between codons 30 and 31 and codons 89 and 90; these are also present in the sheep and bovine sequences. Other amino acid residues identical only in sheep, cattle and m i n k PrPs are leucine at codon 138, valine at codon 184, alanine at codon 230 and isoleucine at codon 233. Three amino acid residues which differ in mink and mouse PrPs are identical in m i n k and hamster PrPs: methionine at codon 113 (codon 108 in the mouse), isoleucine at codon 209 and alanine at
codon 236. Codon 108 in mice with short or intermediate scrapie incubation times encodes leucine; in mice with long incubation times it encodes phenylalanine. In m i n k and hamster PrPs as well as those of sheep and humans it encodes methionine. Recently, Goldgaber (199I) found that the D N A strand opposite to the PrP transcriptional unit contains a large O R F , which for simplicity was n a m e d anti-PrP. There are no stop codons in this large O R F in any h u m a n or animal genome. However, the strand complementary to the mink PrP gene shows numerous stop codons in two reading frames. The third reading frame has a stop signal at the position complementary to codon 249. This codon, which is T C T in human, cattle and sheep PrP genes, and T C C in hamster, rat and mouse PrP genes, is T C A in that of mink. There are a number of possible initiation sites following this stop codon on the complementary strand, although the longest possible O R F follows codon 181. Thus, our sequencing data m a y be an argument against the anti-PrP hypothesis. Should there be a twofold evolutionary pressure on the O R F of the PrP gene, this would apply only to the first 181 codons. The authors wish to thank Professor P. Mehraein, Professor M. Ackenheiland and Dr. D. Wildenauer for their continuing support, as well as S. Hildenbrandt for expert technical assistance, and Drs G. Honold and P. Kufer for their practical help and discussion. This work was supported by research grants from the Wilhelm-Sander-Stiftung (89.036.1) and the Friedrich-Baur-Stiftung to H.A.K.
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